Exhaled Breath Aerosol Shedding of Highly Transmissible Versus Prior Severe Acute Respiratory Syndrome Coronavirus 2 Variants.

BACKGROUND: Aerosol inhalation is recognized as the dominant mode of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. Three highly transmissible lineages evolved during the pandemic. One hypothesis to explain increased transmissibility is that natural selection favors variants with higher rates of viral aerosol shedding. However, the extent of aerosol shedding of successive SARS-CoV-2 variants is unknown. We aimed to measure the infectivity and rate of SARS-CoV-2 shedding into exhaled breath aerosol (EBA) by individuals during the Delta and Omicron waves and compared those rates with those of prior SARS-CoV-2 variants from our previously published work. METHODS: Individuals with coronavirus disease 2019 (COVID-19) (n = 93; 32 vaccinated and 20 boosted) were recruited to give samples, including 30-minute breath samples into a Gesundheit-II EBA sampler. Samples were quantified for viral RNA using reverse-transcription polymerase chain reaction and cultured for virus. RESULTS: Alpha (n = 4), Delta (n = 3), and Omicron (n = 29) cases shed significantly more viral RNA copies into EBAs than cases infected with ancestral strains and variants not associated with increased transmissibility (n = 57). All Delta and Omicron cases were fully vaccinated and most Omicron cases were boosted. We cultured virus from the EBA of 1 boosted and 3 fully vaccinated cases. CONCLUSIONS: Alpha, Delta, and Omicron independently evolved high viral aerosol shedding phenotypes, demonstrating convergent evolution. Vaccinated and boosted cases can shed infectious SARS-CoV-2 via EBA. These findings support a dominant role of infectious aerosols in transmission of SARS-CoV-2. Monitoring aerosol shedding from new variants and emerging pathogens can be an important component of future threat assessments and guide interventions to prevent transmission.

Comparison of Saliva and Midturbinate Swabs for Detection of SARS-CoV-2.

Saliva is an attractive sample for detecting SARS-CoV-2. However, contradictory reports exist concerning the sensitivity of saliva versus nasal swabs. We followed close contacts of COVID-19 cases for up to 14 days from the last exposure and collected self-reported symptoms, midturbinate swabs (MTS), and saliva every 2 or 3 days. Ct values, viral load, and frequency of viral detection by MTS and saliva were compared. Fifty-eight contacts provided 200 saliva-MTS pairs, and 14 contacts (13 with symptoms) had one or more positive samples. Saliva and MTS had similar rates of viral detection (P = 0.78) and substantial agreement (κ = 0.83). However, sensitivity varied significantly with time since symptom onset. Early on (days -3 to 2), saliva had 12 times (95% CI: 1.2, 130) greater likelihood of viral detection and 3.2 times (95% CI: 2.8, 3.8) higher RNA copy numbers compared to MTS. After day 2 of symptoms, there was a nonsignificant trend toward greater sensitivity using MTS. Saliva and MTS demonstrated high agreement making saliva a suitable alternative to MTS for SARS-CoV-2 detection. Saliva was more sensitive early in the infection when the transmission was most likely to occur, suggesting that it may be a superior and cost-effective screening tool for COVID-19. IMPORTANCE The findings of this manuscript are increasingly important with new variants that appear to have shorter incubation periods emerging, which may be more prone to detection in saliva before detection in nasal swabs. Therefore, there is an urgent need to provide the science to support the use of a detection method that is highly sensitive and widely acceptable to the public to improve screening rates and early detection. The manuscript presents the first evidence that saliva-based RT-PCR is more sensitive than MTS-based RT-PCR in detecting SARS-CoV-2 during the presymptomatic period - the critical period for unwitting onward transmission. Considering other advantages of saliva samples, including the lower cost, greater acceptability within the general population, and less risk to health care workers, our findings further supported the use of saliva to identify presymptomatic infection and prevent transmission of the virus.

Infectious Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Exhaled Aerosols and Efficacy of Masks During Early Mild Infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemiology implicates airborne transmission; aerosol infectiousness and impacts of masks and variants on aerosol shedding are not well understood. METHODS: We recruited coronavirus disease 2019 (COVID-19) cases to give blood, saliva, mid-turbinate and fomite (phone) swabs, and 30-minute breath samples while vocalizing into a Gesundheit-II, with and without masks at up to 2 visits 2 days apart. We quantified and sequenced viral RNA, cultured virus, and assayed serum samples for anti-spike and anti-receptor binding domain antibodies. RESULTS: We enrolled 49 seronegative cases (mean days post onset 3.8 ±â€…2.1), May 2020 through April 2021. We detected SARS-CoV-2 RNA in 36% of fine (≤5 µm), 26% of coarse (>5 µm) aerosols, and 52% of fomite samples overall and in all samples from 4 alpha variant cases. Masks reduced viral RNA by 48% (95% confidence interval [CI], 3 to 72%) in fine and by 77% (95% CI, 51 to 89%) in coarse aerosols; cloth and surgical masks were not significantly different. The alpha variant was associated with a 43-fold (95% CI, 6.6- to 280-fold) increase in fine aerosol viral RNA, compared with earlier viruses, that remained a significant 18-fold (95% CI, 3.4- to 92-fold) increase adjusting for viral RNA in saliva, swabs, and other potential confounders. Two fine aerosol samples, collected while participants wore masks, were culture-positive. CONCLUSIONS: SARS-CoV-2 is evolving toward more efficient aerosol generation and loose-fitting masks provide significant but only modest source control. Therefore, until vaccination rates are very high, continued layered controls and tight-fitting masks and respirators will be necessary.

Marek's disease virus oncogene Meq expression in infected cells in vaccinated and unvaccinated hosts.

Marek's disease (MD) vaccines are unique in their capability to prevent MD lymphomas as early as a few days after vaccination, despite the fact that they do not eliminate virulent viruses from the host. To help understand the mechanism behind this unique MD vaccine effect, we compared the expression of MDV oncoprotein Meq among CD4+ T cells between vaccinated and unvaccinated birds. Chickens were vaccinated by an MD vaccine, herpesvirus of turkeys, and then challenged by a recombinant virulent MDV that expresses green fluorescent protein simultaneously with Meq. We found significantly fewer Meq-expressing CD4+ T cells appeared in peripheral blood mononuclear cells (PBMC) of the vaccinated birds compared to the unvaccinated birds as early as one week after the virulent virus challenge. In contrast, the quantity of virulent MDV genome remained similar in Meq- PBMC in both vaccinated and unvaccinated birds. Our results suggest that MD vaccination affects the dynamics of Meq-expressing, possibly transformed, cells while impact on the overall infection in the Meq- cells was not significant.

Viral replication and innate immunity of feline herpesvirus-1 virulence-associated genes in feline respiratory epithelial cells.

Feline herpesvirus-1 (FHV-1) infection occurs worldwide and is a leading cause of respiratory and ocular diseases in cats. Current vaccines reduce the severity of symptoms but do not prevent infection and, therefore, do not provide defense against an establishment of latency and reactivation. We hypothesize that immunomodulation of FHV-1 is the cause of lack in protection and that deletion of virulence/immune modulatory genes of FHV-1 will enhance safety and immunogenicity. Our objective was to use feline respiratory epithelial cell (FREC) cultures to define in vitro growth characteristics and immunomodulation resulting from infection of FRECs with the virulent FHV-1 strain C27 (WT) and glycoprotein C-deletion (gC-), glycoprotein E-deletion (gE-), serine/threonine protein kinase-deletion (PK-), as well as gE and thymidine kinase-double-deletion (gE-TK-) mutants generated by bacterial artificial chromosome mutagenesis. Differentiated FRECs were mock inoculated or inoculated with WT, gC-, gE-, PK-, or gE-TK- mutants. Virus titration and real-time quantitative PCR assays were performed on samples collected at 1 hpi followed by 24 h intervals between 24 and 96 hpi to determine growth kinetics. Real-time PCR was used to quantitate IFNα, TNFα, IL-1ß, IL-10, and TGFß-specific mRNA levels. Immunoassays were performed to measure the protein levels of subsets of cytokines/chemokines secreted by FRECs. Inoculation of FRECs with gE-TK- resulted in significantly lower end-point titers than inoculation with WT or gE-. Both PK- and gC- inoculated FRECs also produced significantly lower end-point titers at 96 hpi than WT. Overall, intracellular virus titers were higher than those of extracellular virus. PCR results for viral DNA paralleled the virus titration results. Further, in contrast to WT inoculation, an increase in IFNα and IL-10 mRNA expression was not observed following inoculation with gE-TK- and PK-, but inoculation with gE-TK- and PK- did result in increased TGFß expression in FRECs compared to responses following infection with WT. Moreover, gE-TK- and PK- blocked the inhibition of IL-8 and neutrophil chemoattractant (KC), which was observed following inoculation with WT. In summary, the results obtained in FRECs may be used to predict the safety and immunogenicity characteristics of these mutants in vivo. Our study highlights the value of the FREC system for studying replication kinetics/immune modulation factors of FHV-1 and screening prospective vaccine candidates before their use in experimental cats.

Expression of Marek's Disease Virus Oncoprotein Meq During Infection in the Natural Host.

Gallid herpesvirus 2 (Marek's disease virus, MDV) causes lymphoproliferative Marek's disease (MD), and is unique among alphaherpesviruses as the viral genome encodes an oncoprotein, Meq. To elucidate the temporal relationship between Meq expression and the development of MD lymphomas in infected chickens, we generated a virulent recombinant MDV that expresses GFP simultaneously with Meq. By using this virus, we monitored the dynamics of Meq expression in vivo throughout the course of infection. In peripheral blood mononuclear cells, the percentage of Meq-expressing cells dramatically increased in the early latent phase but decreased thereafter. Furthermore, we discovered evidences that indicate some of the infected lymphocytes did not express Meq during the latent phase of MDV pathogenesis. These findings provide the first insight into the temporal relationship between Meq expression and MD progression, and new clues to refine the current MD pathogenesis model.

In vitro characterization of felid herpesvirus 1 (FHV-1) mutants generated by recombineering in a recombinant BAC vector.

Felid herpesvirus 1 (FHV-1) mutants were constructed using two-step Red-mediated recombination techniques based on a virulent full-length FHV-1 BAC clone. The individual mutant viruses generated were deficient in glycoprotein C (gC), glycoprotein E (gE), US3 serine/threonine protein kinase (PK), or both gE and thymidine kinase (TK). The gC- mutant virus produced plaques that were similar in size to those resulting from infection with the C-27 parent strain. In contrast, the gE(-), PK(-), and gE(-)PK(-) deletion mutants produced plaques that were significantly smaller. Multistep in vitro growth kinetics of the gE(-), PK(-), and gE(-)PK(-) viruses were slightly delayed compared to those of the C-27 parent strain. Peak progeny titers of these three mutants were approximately 10-fold lower than those generated with the C-27 strain. There was no delay in the growth kinetics of the gC- mutant, but the progeny virus titer obtained with this mutant was at least 3 logs lower compared to the parental strain titer. Based upon their in vitro characteristics, these mutants will be useful for the development of novel immunization strategies against this important feline pathogen.

Difluorosialic acids, potent novel influenza virus neuraminidase inhibitors, induce fewer drug resistance-associated neuraminidase mutations than does oseltamivir.

Neuraminidase inhibitors (NAIs), including the most frequently prescribed oral therapeutic oseltamivir, play a critical role in the control of severe influenza virus (IFV) infections. However, recent reports of spread of an oseltamivir-resistant H1N1 pandemic strain in individuals who have never been exposed to oseltamivir highlight an urgent need for new antivirals against NAI-resistant IFVs. Difluorosialic acids (DFSAs) are a novel class of anti-IFV NAIs designed based on the mechanism of action of IFV NA, and distinguished by their covalent inhibition mode and their high structural similarity to the natural substrate, sialic acid. These characteristics should render the development of resistance a less rapid process. In this report, we evaluated the relative propensity of influenza A virus (IFV-A) NA to develop resistance against the DFSA class of inhibitor by passaging IFV-A strains in vitro in the presence of either oseltamivir or a representative DFSA (FeqGuDFSA). All the passage-selected lines gained mutations in hemagglutinin. Among the 12 oseltamivir-resistant passaged lines, five gained NA mutations and four of these were the well-defined H275Y mutation that causes oseltamivir resistance. In contrast, out of 15 DFSA-passaged lines, only 2 lines gained NA mutations. Further, NA inhibition assays indicated that these mutations did not change the sensitivity of NA to DFSA and thus the resistance to DFSA was not conferred by these NA mutations. These results strongly suggest that, compared to oseltamivir, IFV is less prone to development of resistance against DFSAs through NA mutations.

Dimeric RNA recognition regulates HIV-1 genome packaging.

How retroviruses regulate the amount of RNA genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates genome packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small genome or one copy of a large genome being packaged, whereas regulation by copy number would result in two copies of a genome being packaged independent of size. To distinguish between these two hypotheses, we examined the packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA genome encapsidation not by the mass of RNA but by packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the packaging of viral RNAs containing two packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 genome encapsidation and provide insights into a critical step in the generation of infectious viruses.

Complete genomic sequence and an infectious BAC clone of feline herpesvirus-1 (FHV-1).

Infection with feline herpesvirus-1 (FHV-1) is a major cause of upper respiratory and ocular diseases in Felidae. We report the first complete genomic sequence of FHV-1, as well as the construction and characterization of a bacterial artificial chromosome (BAC) clone of FHV-1, which contains the entire FHV-1 genome and has the BAC vector inserted at the left end of U(L). Complete genomic sequences were derived from both the FHV-1 BAC clone and purified virion DNA. The FHV-1 genome is 135,797bp in size with an overall G+C content of 45%. A total of 78 open reading frames were predicted, encoding 74 distinct proteins. The gene arrangement is collinear with that of most sequenced varicelloviruses. The virus regenerated from the BAC was very similar to the parental C-27 strain in vitro in terms of plaque morphology and growth characteristics and highly virulent in cats in a preliminary in vivo study.